The Microalgae Chlamydomonas for Bioremediation and Bioproduct Production

The extensive metabolic diversity of microalgae, coupled with their rapid growth rates and cost-effective production, position these organisms as highly promising resources for a wide range of biotechnological applications. These characteristics allow microalgae to address crucial needs in the agricultural, medical, and industrial sectors. Microalgae are proving to be valuable in various fields, including the remediation of diverse wastewater types, the production of biofuels and biofertilizers, and the extraction of various products from their biomass. For decades, the microalga Chlamydomonas has been widely used as a fundamental research model organism in various areas such as photosynthesis, respiration, sulfur and phosphorus metabolism, nitrogen metabolism, and flagella synthesis, among others. However, in recent years, the potential of Chlamydomonas as a biotechnological tool for bioremediation, biofertilization, biomass, and bioproducts production has been increasingly recognized. Bioremediation of wastewater using Chlamydomonas presents significant potential for sustainable reduction in contaminants and facilitates resource recovery and valorization of microalgal biomass, offering important economic benefits. Chlamydomonas has also established itself as a platform for the production of a wide variety of biotechnologically interesting products, such as different types of biofuels, and high-value-added products. The aim of this review is to achieve a comprehensive understanding of the potential of Chlamydomonas in these aspects, and to explore their interrelationship, which would offer significant environmental and biotechnological advantages.


Introduction: Why Microalgae and Why Chlamydomonas?
Microalgae represent a broad array of single-celled, photosynthetic organisms that serve as key contributors to primary production across our planet [1].Microalgae can adopt photoautotrophic, heterotrophic, or mixotrophic modes of life, displaying a spectrum of cell sizes, shapes, and structures.Responsible for a significant portion of the global carbon capture, microalgae play a crucial role in supporting ecosystems [2].Microalgae share a common evolutionary origin that can be traced back to a primary endosymbiotic event involving a cyanobacterium, which eventually evolved into the plastid [3].This process has resulted in the emergence of a wide range of colorful and metabolically diverse algal groups, such as diatoms and dinoflagellates [4].
Microalgae are employed in activities such as wastewater treatment [5], biofuel generation [6], animal feed production [7], and the extraction of high-value-added products [8], among other applications.Additionally, microalgae show great potential as organisms for enhancing biological carbon sequestration aimed at mitigating global warming [9].Recently, significant technical advancements, new applications, and products in microalgal biotechnology have been highlighted, showcasing how microalgae can provide high-tech, low-cost, and eco-friendly solutions for current and future societal needs [10].This study also explores how emerging technologies, such as synthetic biology, high-throughput phenomics, and automation, can enhance the understanding of algal biology and drive the development of an algal-based bioeconomy.Consequently, microalgae hold significant ecological and economic potential.
Chlamydomonas is a microalga that is commonly found in freshwater and saltwater habitats, as well as in soil and snow.Taxonomically, the genus Chlamydomonas comprises more than 500 species [11].Over time, it has evolved into a highly influential model organism, thanks to its numerous interesting characteristics [12].Among the Chlamydomonas species, Chlamydomonas reinhardtii is the most commonly used due to its interesting characteristics.Among these features, C. reinhardtii has two flagella, grows well in axenic cultures, exhibits a relatively rapid doubling time of approximately 8-12 h, and its nuclear, chloroplast, and mitochondrial genomes are sequenced.Additionally, C. reinhardtii exhibited an exceptional ability to adapt and thrive under nearly all experimental conditions tested in heterotrophic, phototrophic, and mixotrophic cultivations [13].Moreover, the Chlamydomonas Sourcebook [14] provides a thorough overview of essential research areas, historical background, physiology, and methodologies related to Chlamydomonas.Additionally, the Chlamydomonas Resource Centre offers a wide range of resources, including biochemical assays, protocols, plasmids, and a diverse collection of mapped mutant strains.Chlamydomonas biotechnology has centered on finding high-yielding strains through exploration of natural sources and improving productivity through forward genetics.Recent progress has been made in high-throughput screening, genome-wide mutant libraries, and genome editing techniques with Chlamydomonas [15].Furthermore, enhancing the yield of many biotechnological processes involving Chlamydomonas can be achieved through synergistic interactions with other microorganisms, predominantly bacteria [16,17].
However, there are still numerous challenges hindering the efficient utilization of Chlamydomonas biotechnologically in bioremediation and bioproduct production.Consequently, substantial efforts are being directed towards gaining a deeper understanding of the biological mechanisms relevant to its applications.To the best of our knowledge, there has never been a single comprehensive review covering all these aspects of Chlamydomonas.Therefore, here we summarize and categorize these reports with the aim of highlighting the potential of Chlamydomonas to fulfill these tasks.

Wastewater and Advantages of Using Microalgae for Its Bioremediation
Wastewater comprises a diverse mixture of organic and inorganic compounds, as well as synthetic substances that reflect societal lifestyles and technology.Carbohydrates, fats, sugars, and amino acids are among the primary contaminants found in wastewater.Indeed, amino acids constitute three-quarters of the organic carbon in some wastewater [18].Inorganic constituents found in wastewater include a variety of substances such as calcium, sodium, magnesium, potassium, sulfur, arsenic, bicarbonate, heavy metals, nitrates, chlorides, phosphates, and non-metallic salts [19].Persistent organic pollutants include chlorinated and aromatic compounds, such as polychlorinated biphenyls, polycyclic aromatic hydrocarbons, and organochlorine pesticides [20].The composition of wastewater varies depending on its source.Municipal wastewater is generated from households, commercial establishments, and institutions.It typically contains organic matter, nutrients, pathogens, and various chemicals from soaps and detergents [21].Agricultural wastewater originates from farming activities and can contain organic matter, pesticides, herbicides, and fertilizers [22].Industrial wastewater may include a diverse array of industry-specific pollutants, including heavy metals, organic chemicals, and oils [23].Each type of wastewater has its own unique characteristics and requires specific treatment approaches to address its particular contaminants.
As anthropogenic activities increase, resulting in more complex wastewater compositions, it becomes crucial to develop wastewater treatment procedures that are easy to implement, efficient, and environmentally friendly.Traditional methods for treating wastewater include physical, mechanical, chemical, and biological approaches (Figure 1).Physical methods entail processes such as sedimentation, screening, and skimming, while mechanical methods include filtration techniques like ceramic membrane and sand filter technology [24].Chemical methods involve processes such as neutralization, adsorption, precipitation, disinfection, and ion exchange [25].However, purely physical-chemical methods have proven ineffective in treating wastewater with complex compositions.Biological methods for wastewater treatment involve the use of microorganisms that consume pollutants in the wastewater as food [26].However, biological wastewater treatment also has various drawbacks, including high energy consumption, expenses associated with aeration, and challenges in sludge management.Therefore, the integration of physical-chemical and biological methods is an effective approach for sustainable wastewater treatment [27].
Cells 2024, 13, x FOR PEER REVIEW 3 of 28 wastewater include physical, mechanical, chemical, and biological approaches (Figure 1).Physical methods entail processes such as sedimentation, screening, and skimming, while mechanical methods include filtration techniques like ceramic membrane and sand filter technology [24].Chemical methods involve processes such as neutralization, adsorption, precipitation, disinfection, and ion exchange [25].However, purely physical-chemical methods have proven ineffective in treating wastewater with complex compositions.Biological methods for wastewater treatment involve the use of microorganisms that consume pollutants in the wastewater as food [26].However, biological wastewater treatment also has various drawbacks, including high energy consumption, expenses associated with aeration, and challenges in sludge management.Therefore, the integration of physicalchemical and biological methods is an effective approach for sustainable wastewater treatment [27].Phycoremediation (where 'phyco' means algae in Greek) is a sustainable and environmentally friendly approach that utilizes various types of algae, including cyanobacteria, microalgae, and macroalgae, to remove or extract pollutants from wastewater (Figure 1).Among the benefits of phycoremediation are the removal of nutrients and xenobiotic substances, the reduction in excess nutrients from effluent with high organic material, CO2 mitigation, the treatment of effluents with heavy metal ions, and the monitoring of potentially toxic substances using algae as biosensors [28].Microalgae have the ability to absorb and break down contaminants through processes such as biosorption, bioaccumulation, and biotransformation [29].Phycoremediation not only helps in the removal of pollutants but also results in the production of algal biomass, which can be utilized for various valuable products such as food, feed, fertilizers, pharmaceuticals, and biofuels [30].A wide range of non-pathogenic algae are utilized for wastewater treatment, such as Chlorella sp., Spirulina sp., Scenedesmus sp., Nostoc sp., and Oscillatoria sp., [31].In this review, we will focus on those studies that use Chlamydomonas in phycoremediation.

Microalgae Cultivation Methods
Microalgae cultivation methods are categorized into suspended systems (including open reactors and closed photobioreactors) and attached systems, such as biofilm reactors (Figure 1).Open reactors include lakes and natural ponds, as well as specially designed high-rate algal ponds (HRAPs) that are tanks or lagoons featuring a paddle wheel that Phycoremediation (where 'phyco' means algae in Greek) is a sustainable and environmentally friendly approach that utilizes various types of algae, including cyanobacteria, microalgae, and macroalgae, to remove or extract pollutants from wastewater (Figure 1).Among the benefits of phycoremediation are the removal of nutrients and xenobiotic substances, the reduction in excess nutrients from effluent with high organic material, CO 2 mitigation, the treatment of effluents with heavy metal ions, and the monitoring of potentially toxic substances using algae as biosensors [28].Microalgae have the ability to absorb and break down contaminants through processes such as biosorption, bioaccumulation, and biotransformation [29].Phycoremediation not only helps in the removal of pollutants but also results in the production of algal biomass, which can be utilized for various valuable products such as food, feed, fertilizers, pharmaceuticals, and biofuels [30].A wide range of non-pathogenic algae are utilized for wastewater treatment, such as Chlorella sp., Spirulina sp., Scenedesmus sp., Nostoc sp., and Oscillatoria sp., [31].In this review, we will focus on those studies that use Chlamydomonas in phycoremediation.

Microalgae Cultivation Methods
Microalgae cultivation methods are categorized into suspended systems (including open reactors and closed photobioreactors) and attached systems, such as biofilm reactors (Figure 1).Open reactors include lakes and natural ponds, as well as specially designed high-rate algal ponds (HRAPs) that are tanks or lagoons featuring a paddle wheel that circulates wastewater.HRAPs can be an economical and sustainable method for treating wastewater, as microalgae efficiently absorb nutrients such as phosphorus and nitrogen, as well as help remove organic and inorganic contaminants [32].Closed photobioreactors (PBR) are enclosed systems utilized for the cultivation of microalgae and other phototrophic microorganisms.They provide excellent control over culture conditions with minimal risk of contamination.Different types of PBRs include flat panel, tubular, and stirred tank designs [33].The cultivation of microalgae in biofilm reactors involves immobilizing the microalgae on a surface that acts as a support, forming a continuous layer.This method offers advantages such as higher concentration per unit volume of medium, reduction or absence of cells in the effluent, and ease of harvesting [34].The extraction and dewatering of algae cells from biofilms are simplified by the ease of separating attached cells from their growth medium.In the context of technological applications, regulating the adhesion properties of Chlamydomonas could significantly enhance the efficiency of biofilm reactors by controlling surface colonization and biofilm formation.So far, the basic principles governing the colonization of surfaces by motile, photosynthetic microorganisms remain largely unexplored.Interestingly, Chlamydomonas has the ability to secrete substances such as sulphated polysaccharides that act as antibiofilm agents for certain bacteria, preventing these bacteria from attaching to the biofilm [35].This property can be highly beneficial in controlling the occurrence of bacterial contaminations.

Chlamydomonas Phycoremediation
Microalgae, particularly Chlamydomonas, exhibit a remarkable capacity and diversity in bioremediating various molecules.Next, we will present the main mechanisms for bioremediation.Biosorption is a passive mechanism whereby microalgae serve as a biological sorbent to capture and accumulate pollutants.Microalgae utilize their cell wall and various chemical groups to attract and retain contaminants [36].Microalgae can remove pollutants through bioaccumulation.The main differences between biosorption and bioaccumulation processes lie in their mechanisms.Biosorption is a passive process where microorganisms utilize their cellular structure to capture pollutants on the binding sites of the cell wall.On the other hand, bioaccumulation is an active process that involves the accumulation of pollutants in the biomass of microalgae, either by accumulation or uptake into intracellular spaces [37].Bioaccumulation requires cellular growth and is typically slower than biosorption.Biotransformation involves the breakdown of pollutants, either inside or outside the cells, facilitated by enzymes [38].While there are not significant concerns with biosorption and bioaccumulation, biotransformation presents more challenges due to the possibility of its products being potentially more toxic than the original compounds.
Some studies have cultivated Chlamydomonas in PBRs for the decontamination of wastewater [39] (Table 1).In this regard, Chlamydomonas debaryana using dairy wastewater reduced nitrogen, phosphorus, organic carbon, and chemical oxygen demand by more than 85% [40].C. debaryana and C. reinhardtii were able to effectively treat swine wastewater [41].With C. reinhardtii, 55.8 mg of nitrogen and 17.4 mg of phosphorus per liter per day were effectively removed from industrial wastewater [42].Using C. mexicana, a high removal efficiency of nitrogen (62%), phosphorus (28%), and inorganic carbon (29%) was achieved in piggery wastewater [43].Research shows that nitrogen-limited wastewater microalgae can be effectively used for biomass production through anaerobic fermentation [44].Wastewater collected from a paper industry was treated using C. reinhardtii, resulting in significant reductions in nitrate (86%), phosphate (88%), and chemical oxygen demand (COD) (93%) [45].
Numerous studies have reported the use of HRAP in wastewater treatment, primarily focusing on genera such as Scenedesmus and Chlorella [46].However, very few records exist of applying HRAP with Chlamydomonas.In a pilot-scale HRAP experimental wastewater treatment, Chlamydomonas sp. was found to be one of the dominant genera.The study reported a reduction in the biochemical oxygen demand by 90%, chemical oxygen demand by 65%, total nitrogen by 46%, and total phosphorus by 20% [47].A study on the bioremediation of piggery wastewater using HRAP revealed that Chlamydomonas sp. was the dominant species, with average chemical oxygen demand and total nitrogen removal efficiencies of 76% and 88%, respectively [48].In another study employing HRAP with Chlamydomonas sp. for treating municipal wastewater, average reductions in volatile suspended solids, total nitrogen, and biochemical oxygen demand were 63%, 76%, and 98%, respectively [49].Chlamydomonas sp.JSC4 has been successfully employed in a biofilm reactor for the removal of phosphorus, nitrogen, and copper from swine wastewater [50].In a biofilm reactor, Chlamydomonas pulvinata TCF-48 g has demonstrated significant polyphosphate accumulation and a high phosphorus removal rate of 70%, making it valuable for phosphate recovery applications [51].The encapsulation of C. reinhardtii in alginate beads has been successfully carried out to remove various types of contaminants such as phosphorus, nitrogen, lead, mercury, and cadmium [52] or even phenol [53].
Chlamydomonas can metabolize xenobiotics through a wide range of enzymatic processes, including CYP450 oxidation reactions, hydrolysis, glutamate conjugation, and methylation [82].Chlamydomonas moewusii excretes laccases capable of breaking down and detoxifying phenolic pollutants [83].The toxicity responses of different pollutants, such as benzophenone-3, bisphenol A, oxytetracycline, and atrazine, in C. reinhardtii showed a similar pattern: an increase in chlorophyll autofluorescence and a decrease in growth rate and vitality [84].The biotransformation of five bisphenol derivatives (AF, B, F, S, and Z) by C. mexicana shows that all the biotransformed products were less toxic than the parent compounds [85].Chlamydomonas has also been used in efforts to degrade commonly used plastic components such as Polyethylene terephthalate (PET).In Ideonella sakaiensis, a novel plastic degradation enzyme called PETase has been identified [86].The I. sakaiensis PETase has been expressed through genetic recombination in the C. reinhardtii nucleus and chloroplast genomes, showing a significant ability to break down PET [87].Under specific adverse conditions, such as NaCl stress, EDTA exposure, or acidic pH, C. reinhardtii can form multicellular aggregates called palmelloids.These are small clonal structures that result from cells failing to separate after division [88].The defense mechanisms of C. reinhardtii under perchlorate stress were investigated, revealing palmelloid formation when exposed to 100 and 200 mM perchlorate [89].These researchers highlight the metabolic versatility of Chlamydomonas in dealing with xenobiotic compounds, demonstrating its ability to transform and process a variety of chemicals through different mechanisms.The encapsulation of microalgae is a process in which the microalgae are coated with a protective layer to enhance their stability, protect them from adverse conditions, and facilitate their application.This process offers various biotechnological advantages, such as protecting the formation of bioactive compounds, promoting release control, improving solubility, and enhancing bioavailability [90].Various materials, including alginate, carrageenan, chitosan, and polyvinyl, have been used for the immobilization of microalgae [91].In the case of Chlamydomonas, alginate has been the most successful and currently the most commonly used material for encapsulation.The pore size of alginate beads in C. reinhardtii is critical, with the highest efficiency for contaminant removal obtained in gel beads with a pore size of 3.5 mm [92].In this regard, Chlamydomonas cells immobilized with carboxymethyl cellulose beads have demonstrated a great capacity for decontaminating Uranium (VI) through biosorption [93].
Microalgae have been actively employed in initiatives focused on reducing CO 2 emissions due to their ability to absorb CO 2 via photosynthesis.C. reinhardtii exhibits a superior ability to fix CO 2 compared to other photosynthetic organisms [94].Bio-fixation refers to the process by which certain organisms, such as microalgae, utilize CO 2 from the air or other sources like flue gas streams to create biomass.The production of 1 g of microalgae biomass leads to the sequestration of 1.8 g of CO 2 [95].In Chlamydomonas the expression of a single H + -pump increase its tolerance to high concentrations of CO 2 , such as those found in industrial flue gas [96].These findings illustrate the potential of C. reinhardtii to mitigate CO 2 emissions from industrial sources.
The studies mentioned regarding bioremediation with Chlamydomonas present several limitations that we will now outline, which we believe could be addressed in future research.Many studies are conducted at the laboratory or pilot scale.For practical application, it is crucial to evaluate the effectiveness of Chlamydomonas under real conditions, such as in large-scale wastewater treatment plants.Studies often focus on a single species or strain of Chlamydomonas.Investigating a broader range of species and strains would be beneficial to better understand their bioremediation potential.The interaction of Chlamydomonas with other microbial species in the natural environment can impact its efficacy.Studying these interactions and their impact on bioremediation is essential.Some studies mention the biotransformation of contaminants, but the toxicity of the resulting products is not always evaluated.Investigating the effects of these transformed products is critical.Cultivation parameters (such as light, temperature, pH, and nutrient concentration) can affect the efficiency of Chlamydomonas.Optimizing these conditions could improve outcomes.Many studies are short-term.Researching the long-term stability and efficacy of Chlamydomonas in wastewater treatment systems is essential.In summary, improving these studies requires a combination of more realistic experimental approaches, species diversity, toxicity evaluation, and cultivation-condition optimization.These efforts will contribute to a more effective application of Chlamydomonas in bioremediation.

Biomass
One of the main products derived from the cultivation of microalgae is their biomass, as it is used as raw material for obtaining other derived bioproducts.It would be highly beneficial economically to use the biomass resulting from the bioremediation process for bioproduct purification.However, the utilization of biomass derived from wastewater treatment encounters several inherent challenges.These challenges include the scalability of biomass production, the presence of xenobiotics and heavy metals, as well as the contamination with bacteria, fungi, and viruses, all of which limit their extensive application [97].Although numerous efforts are being made to address this issue, the production of the main bioproducts obtained from microalgae still does not use wastewater as a cultivation source.Next, we will present studies that utilize Chlamydomonas to obtain certain bioproducts, some of which use biomass derived from wastewater remediation.
The composition of biomass is influenced by the strains of microalgae and the culture conditions [98].One straightforward method to increase biomass productivity involves altering the culture medium conditions or adjusting the supply of certain macroelements.For example, in microalgae, some researchers have evaluated the effect of various carbon sources [99], pH variations [100], and photoperiods [101], as well as trace element compositions for biomass production [102].Consequently, various approaches have been explored to optimize microalgae biomass enriched in specific biomolecules (Figure 2).The highest biomass concentration of Chlamydomonas obtained so far has been heterotrophically with acetate, reaching 23 g/L [103] (Table 2), far behind other green algae that are able to consume glucose as a substrate, like Chlorella sp. and Scenedesmus sp., for which biomass reached 271 g/L and 286 g/L, respectively [104].Chlamydomonas cells can be cultivated in various conditions.After cultivation, the collected biomass and medium can then be processed as indicated to obtain the specified bioproducts (see Table 2).

Biochar
Biochar is a carbonaceous material produced through the pyrolysis of biomass (Figure 2), which can be obtained from microalgae, agricultural residues, wood, or organic waste [105].Biochar is characterized by its high porosity and specific surface area, making it useful for improving soil quality and carbon sequestration.It is used in agriculture as a Figure 2. The process of Chlamydomonas bioproduct generation.Chlamydomonas cells can be cultivated in various conditions.After cultivation, the collected biomass and medium can then be processed as indicated to obtain the specified bioproducts (see Table 2).

Biochar
Biochar is a carbonaceous material produced through the pyrolysis of biomass (Figure 2), which can be obtained from microalgae, agricultural residues, wood, or organic waste [105].Biochar is characterized by its high porosity and specific surface area, making it useful for improving soil quality and carbon sequestration.It is used in agriculture as a soil amendment to enhance soil structure, retain nutrients and water, and promote beneficial microbial activity.Additionally, biochar is considered a strategy for mitigating climate change, as burying it in the soil can store carbon stably for long periods [106].C. reinhardtii biomass has been successfully used to prepare biochar [107].The highest biochar yield was 93.9%, achieved through dry torrefaction at 200 • C using Chlamydomonas sp.JSC4 [108].Biochar prepared from Chlamydomonas sp. has been shown to have a high capacity for removing contaminants [109] (Table 2).

Biofertilizers
Microalgae are used as a biofertilizers and biostimulants by promoting crop growth and increasing soil nutrient contents, thereby reducing the usage of chemical fertilizers [110].In contrast, Chlamydomonas species have received little attention and are not fully utilized in agriculture, despite being among the most abundant microalgae species in natural soil ecosystems.In this regard, a study on the effects of Chlamydomonas applanata M9V as a biofertilizer on wheat found that it performed even better than a certain amount of chemical fertilizer [111].Acid-hydrolyzed dry biomass of C. reinhardtii improved the phosphorus, nitrogen, and carotenoid contents of Solanum lycopersicum [112].The application of live Chlamydomonas cells significantly increased leaf size, shoot length, fresh weight, number of flowers, and pigment content of Medicago truncatula [113].Lyophilized powders derived from C. reinhardtii have been found to positively affect the growth of maize plants by producing bioactive compounds that act as biostimulants, enhancing plant growth, crop performance, yields, and quality [114].Biomass extracts of Chlamydomonas sp.exhibited auxin-like activity that increased the number of roots in cucumber plants [115].Chlamydomonas sajao can improve soil physical properties, such as aggregation and stability, thereby contributing to enhanced soil structure and nutrient retention [116].These results suggest that Chlamydomonas can be an effective alternative to chemical fertilizers for promoting crop growth and yield (Table 2).

Bioplastic
Bioplastics are biodegradable materials derived from renewable biomass sources, offering a sustainable alternative to traditional plastics [117].Various molecules can be used as building blocks for bioplastics, including polyhydroxybutyrate (PHB), starch, TAG, lactic acid, or polybutylene succinate.PHB can be naturally synthesized by certain bacteria, such as Azotobacter or Pseudomonas.PHB production involves three key enzymes: β-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase, encoded by phbA, phbB, and phbC, respectively.Research has focused on engineering Chlamydomonas strains to enhance PHB de novo biosynthesis, as Chlamydomonas naturally cannot synthesize PHB.With this aim, the phbB and phbC genes from Ralstonia eutropha have been inserted into the C. reinhardtii genome, leading to the observation of PHB granules in the cytoplasm [118] (Table 2).While cytosolic accumulation of PHB in Chlamydomonas often results in impaired cell growth and low yield, peroxisomes have emerged as a promising alternative.A complete PHB biosynthesis pathway has been successfully reconstructed by expressing the three PHB synthesis genes and targeting the proteins to the peroxisomes.Within the peroxisomes of these strains, PHB reached 21.6 mg/g, which represents a 3600-fold increase over cytosolic PHB production [119].Another strategy is to use TAG as the building block for bioplastics.TAG synthesized by C. reinhardtii has been directly crosslinked with glycerol or ammonium persulfate and molded into plastic beads that are capable of withstanding compressive stress up to 1.7 megapascals [120].Cell-plastics are a type of bioplastic that directly utilizes raw cells and the hydrolyzed cell broth.Unlike conventional bioplastics, cell-plastics do not require exhaustive processes for extracting and refining the biomolecules that serve as the building blocks.Recently, Chlamydomonas cells have arisen as the constituent blocks of this new type of bioplastic, as their cell size and protein-rich, cellulose-free cell wall were demonstrated to be ideal components for its fabrication [121].

Biofuels
Biofuels are fuels derived from renewable biological sources such as plants or plantderived materials.First-generation biofuels are produced from food crops.Secondgeneration biofuels are derived from non-food sources such as waste, and third-generation biofuels are produced from sources that do not compete with arable land, such as microalgae [122].Microalgae have regained attention as alternative resources for environmentally friendly production of biofuels, including biodiesel, bioethanol, biogas, and biohydrogen.These biofuels can be produced through thermochemical and biochemical conversions, photosynthesis-mediated microbial fuel production, and transesterification [123].

Biodiesel
Triacylglycerols (TAG) are crucial lipids in microalgae for biofuel production.Oleaginous microalgae, rich in TAG, can be converted into biodiesel through transesterification, a process that transforms TAG into fatty acid methyl esters, the key components of biodiesel [124].Utilizing Chlamydomonas sp.JSC4, a direct transesterification process was employed, resulting in nearly 100% biodiesel production in a single step [125] (Table 2).Given that biodiesel production is closely linked to the quantity of lipids and TAGs, various strategies have been explored to enhance their production in Chlamydomonas.Some studies have focused on elucidating the functions of key genes involved in lipid and TAG production.The down-regulation of the phosphoenolpyruvate carboxylase gene in C. reinhardtii resulted in a 74.4% increase in lipid content [126].The overexpression of acetyl-CoA synthetase resulted in a 2.4-fold increase in the accumulation of TAG [127].In C. reinhardtii, the mutation of ACX2, which encodes a member of the acyl-CoA oxidase responsible for the first step of peroxisomal fatty acid beta-oxidation, resulted in an accumulation of 20% more lipid [128].A mutant of C. reinhardtii deficient in phospholipase showed an increase in TAG content of up to 190% [129].The overexpression of the ferredoxin gene PETF in C. reinhardtii resulted in higher lipid content [130].The Target of Rapamycin (TOR) plays a crucial role in regulating cell growth.It has been shown that mutants of C. reinhardtii lacking TOR experience an increase in TAG production [131].The strategy of heterologously overexpressing genes in Chlamydomonas has been successful in increasing TAG content.In this sense, the heterologous expression of the Dunaliella tertiolecta fatty acyl-ACP thioesterase in C. reinhardtii leads to increased lipid production [132].By expressing the diacylglycerol acyltransferase from Saccharomyces cerevisiae into C. reinhardtii, the fatty acids and TAG content increased by 22% and 32%, respectively [133].The heterologous expression of Lobosphaera incisa glycerol-3-phosphate acyltransferase in C. reinhardtii enhances TAG production [134].The synthesis of starch and lipids competes for carbon skeletons; thus, inhibiting starch synthesis is another strategy for increasing TAG production.In this sense, silencing ADP-glucose pyrophosphorylase in C. reinhardtii resulted in a tenfold increase in TAG content [135].Genetically modifying Chlamydomonas sp.JSC4 in the gene that encodes the starch debranching enzyme promotes carbohydrate degradation and redirects carbon resources into lipids, resulting in a 1.46-fold increase in lipid content [136].
A commonly employed approach to accumulate TAG in Chlamydomonas is to induce stress conditions, particularly nutrient limitation or starvation [137].C. reinhardtii exhibits a notable increase in TAG accumulation under low nitrogen concentrations [138].Under nitrogen deprivation, C. reinhardtii starch mutants exhibit almost a 10-fold increase in TAG [139].Under nitrogen limitations, increasing the expression of S-adenosylmethionine synthetase in C. reinhardtii enhances cell viability and TAG production [140].Phosphorus stress also triggers TAG production in Chlamydomonas [141].Additionally, a higher TAG content is generated under conditions of low sulfur concentration [142].The lipid content in C. mexicana was observed to rise as the concentration of NaCl was increased to 25 mM [143].The lipid content of the C. reinhardtii starchless mutant BAF-J5 increased by 76% following a temperature shift to 32 • C [144].
Increasing TAG levels by inducing stress conditions often comes at the expense of inhibited microalgal growth.Under these conditions, there is an inverse relationship between TAG yield and microalgal growth.To mitigate this, it has been reported that overexpressing the transcription factor MYB1 in C. reinhardtii, which mediates lipid accumulation, results in nearly 60% more TAG without negatively impacting cell growth [145].In another strategy, a cultivation approach involving two stages has been proposed, wherein C. reinhardtii experiences nutrient stress only after an initial period of optimal growth, allowing for high TAG accumulation [146].The development of effective methods for cultivating Chlamydomonas is essential in biodiesel production.In this regard, in C. reinhardtii, a multi-parametric kinetic model developed using computational tools has been proven, resulting in significant increases in lipids (74%) [147].

Bioethanol
Bioethanol is a biofuel that can be obtained through the fermentation of various types of biomass containing high amounts of sugars.For bioethanol production, the high carbohydrate content present in both the cellulose and hemicellulose cell walls, as well as the starch-based cytoplasm, is broken down into monomeric sugars during enzymatic hydrolysis prior to fermentation.However, the cell wall of Chlamydomonas is not made of cellulose like in plants, but of five dense, glycoprotein-rich layers [148].Therefore, efforts have been focused on utilizing starch-rich Chlamydomonas for the production of bioethanol.The biomass of C. reinhardtii UTEX 90 was converted into glucose through two hydrolytic steps using α-amylase and amyloglucosidase, with nearly all the starch successfully transformed into glucose without damaging the cell wall, reducing the costs of bioethanol purification [149] (Table 2).Pretreating C. reinhardtii UTEX 90 biomass with sulfuric acid (1-5%) at temperatures ranging from 100 to 120 • C significantly increases the glucose release for the production of bioethanol [150].The supraoptimal temperature treatment method, which involved cultivating C. reinhardtii at 39 • C despite its optimal temperature being 25 • C, was successfully applied and resulted in nearly a threefold enhancement of starch content [151].The hormones have also been described to have a very important role in starch accumulation; in this sense, in Chlamydomonas, 100 µM of Indole-3-acetic acid produces an accumulation of up to nine-times more starch [152].Chlamydomonas sp.QWY37 has been effectively utilized for bioethanol production from swine wastewater, achieving a maximum bioethanol yield of 61 g/L [153].

Biogas
Biogas is a renewable energy source primarily composed of CH 4 , derived from the microbial anaerobic digestion of biomass obtained from various sources (Figure 2).The production of biogas involves multiple stages, including hydrolysis, acidogenesis, acetogenesis, and methanogenesis, which are facilitated by a microbial consortium that plays a crucial role in influencing both the composition and yield of the biogas [154].This process eliminates the need to extract specific macromolecules, such as lipids, proteins, or carbohydrates, and can be carried out using wet biomass [155].The fermentation of C. reinhardtii biomass produces approximately 587 mL of biogas per gram of volatile solids [156].However, microalgae biomass is not ideal for biogas generation due to its high protein content, which results in an unfavorably low carbon-to-nitrogen ratio.This imbalance arises because the ammonia released during protein degradation inhibits the methanogenesis process [157].C. reinhardtii biomass has been studied for its potential in overcoming this limitation.In this regard, the anaerobic digestion of C. reinhardtii biomass obtained in low-nitrogen media has shown remarkable efficiency in biogas production due to its high carbon-to-nitrogen ratio [158] (Table 2).
The high resistance of microalgae biomass to microbial decomposition due to their rigid cell walls is a significant challenge in biogas production.However, since the main components of the C. reinhardtii cell wall are glycoproteins rather than cellulose, C. reinhardtii has been shown to produce larger quantities of biogas compared to species with more complex cell walls (such as Chlorella sp. and Scenedesmus sp.) [159].The findings revealed that the C. reinhardtii cell wall was not an obstacle but instead became advantageous by enabling the gradual degradation of intracellular content [160].One way to valorize the microalgal biomass produced during wastewater treatment is to utilize it as a source for biogas production, thereby reducing the economic costs of treatment [161].In this regard, Chlamydomonas sp.Ck has demonstrated high efficiency in decontaminating piggery wastewater while simultaneously producing a high biogas yield [44].For all the reasons mentioned, anaerobic digestion of C. reinhardtii biomass can be considered a cost-effective alternative for biogas production compared to other methods.

Hydrogen
The production of the preceding bioproducts shares the common step of first obtaining biomass, and then extracting these compounds from it.Next, we will present some products that Chlamydomonas releases into the culture medium and therefore can be purified without needing to be extracted from the biomass, thereby reducing the economic cost of their production (Figure 2).A prominent example of this is hydrogen, which has emerged as one of the most promising energy carriers for future energy demands.Hydrogen presents the opportunity to cultivate living organisms such as bacteria, cyanobacteria, and microalgae capable of releasing H 2 into the media [162].Hydrogen is generated through enzymes known as hydrogenases [163].Chlamydomonas has two hydrogenases that have been extensively studied with the aim of increasing their production efficiency [164].The hydrogenases catalyze the reduction of protons into H 2 either using energy from light (biophotolysis) or by oxidizing organic compounds such as starch (dark fermentation).One of the primary biotechnological challenges of using Chlamydomonas as a factory to produce H 2 is the rapid inactivation of its hydrogenases by oxygen, particularly considering that oxygen is generated during photosynthesis.Therefore, the initial evidence indicating that Chlamydomonas was capable of producing H 2 was observed with Chlamydomonas moewusii under anaerobic condition [165], and subsequently with C. reinhardtii, also anaerobically [166].The first successful strategy demonstrating significant and consistent H 2 production under aerobic conditions involved using sulfur-starved C. reinhardtii [167].The reason for this is that the absence of sulfur blocks protein synthesis, thereby halting photosynthesis and oxygen production.Alternative strategies for H 2 production under non-stress conditions are also possible, particularly in media containing acetate, which is compatible with Chlamydomonas growth [168,169].However, the rates of H 2 production under non-stress conditions are lower compared to those under stressful conditions [170].
In Chlamydomonas, numerous genetically engineered strains have been developed to enhance H 2 production.One of the most successful approaches has been to improve the intrinsic oxygen tolerance of hydrogenase through mutagenesis [171].A production of 1200 mL of H 2 per liter has been reported after 6 days using the Photosystem I (PSI) cyclic electron transport mutant pgr5, which is defective in thylakoid proton gradient regulation [172].Another strategy is diverting electron flow to the hydrogenase [173], and degrading or inhibiting the function of Photosystem II (PSII) to prevent oxygen production [174] (Table 2).However, strategies that do not degrade PSII appear to be advantageous, as the long-term loss of PSII inhibits cell growth.In this sense, a PSI-hydrogenase chimera was created by inserting the HydA sequence into the PsaC (stromal subunit of PSI).This redirects photosynthetic electron flow towards proton reduction [175].A disadvantage in the use of Chlamydomonas is that the hydrogen production rate is influenced by the size of microalgae cells.The hydrogen production rate of Chlorella is higher than that of Chlamydomonas due to its relatively smaller size [176].

High-Value Bioproducts
The term "high-value bioproducts" refers to a wide range of products derived from various sources, which economically have a higher value compared to low-to mediumvalue products.C. reinhardtii is a promising organism for the production of high-value bioproducts [177].Glycolate, a high-value cosmetic ingredient, can be overproduced in Chlamydomonas.When Chlamydomonas is in an environment with low CO 2 (0.04%), rubisco oxygenates ribulose-1,5-bisphosphate instead of carboxylating it, consequently producing glycolate.In Chlamydomonas, glycolate is toxic, prompting an active system to excrete it.To facilitate the recovery of potentially lost carbon, the genes for photorespiratory metabolism are induced.Photorespiration detoxifies and recycles glycolate, generating glycerate and releasing CO 2 .In Chlamydomonas, glycolate dehydrogenase (GDH) is involved in photorespiration by oxidizing glycolate to glyoxylate.It has been observed that Chlamydomonas GDH mutants over-accumulate glycolate in the media [178].Chlamydomonas has a CO 2 -concentrating mechanism (CCM) to prevent the rubisco oxygenation reaction and, consequently, glycolate excretion [179].CIA5 is the primary transcription factor that induces the CCM, and its mutation has been shown to increase the amount of excreted glycolate [180].By incorporating 6-Ethoxy-2-benzothiazolesulfonamide (EZA), a CCM inhibitor, glycolate production can be maximized without compromising cell viability.Under these conditions, glycolate accumulates in the medium, reaching a concentration of up to 41 mM [181].In photorespiration, hydroxypyruvate is converted to glycerate by hydroxypyruvate reductase (HPR).In C. reinhardtii, the mutation of hpr1 results in increased excretion of glycolate into the medium [182] (Table 2).
Bioisoprenoids are natural compounds synthesized by plants, animals, and microorganisms through the isoprenoid biosynthetic pathway.These compounds are structurally and functionally diverse, with a wide range of applications, including their use as perfumes, cosmetics, pigments, medicines, and chemical signals.Bioisoprene production has gained attention due to its sustainability and efficiency compared to petrochemical sources [183].It has been demonstrated that C. reinhardtii can be genetically modified to produce significant amounts of bioisoprene by overexpressing four different plant isoprene synthase genes (IspS), with the strain expressing the Ipomoea batatas IspS gene showing the highest isoprene levels [184] (Table 2).
Hydroxyalkanoyloxyalkanoates (HAA) are a type of lipidic surfactants that can be produced by certain bacteria that show great potential for a wide range of applications.They are synthesized by the condensation of hydroxyalkanoic acids, which are produced by the metabolism of fatty acids.The chloroplast genome of C. reinhardtii was engineered by inserting the gene encoding the acyltransferase of P. aeruginosa, a key enzyme in HAA synthesis, resulting in high concentrations of HAA not only in the intracellular fraction but also in the extracellular [185].
There is strong interest in developing bio-based hydrocarbons and their unsaturated analogs, the alkenes, as potential substitutes for hydrocarbons derived from petroleum.The alkene 7-heptadecene has high demand for various biotechnological processes.While the biological function of alkenes in microalgae remains completely unknown, it has been shown that in C. reinhardtii, the enzyme fatty acid photodecarboxylase is responsible for synthesizing 7-heptadecene [186] (Table 2).This discovery opens the possibility of overproducing this alkene in C. reinhardtii.ε-Polylysine is a biodegradable polymer composed of 25-30 lysine monomers that has a variety of applications, including antimicrobial activity and anticancer agent [187].It has been reported that ε-polylysine is produced from Chlamydomonas sp.supplemented with lysine, aspartate, and tricarboxylic acids, achieving a maximum production of 2.24 g/L [188].
Bio-polyamides, also known as nylons, are sustainable polymers derived from renewable resources.Bio-polyamides have excellent material properties, leading to a high demand for polyamide plastics with diverse applications across various industries [189].Cadaverine and putrescine are polyamines commonly used as precursors and building blocks for the synthesis of bio-polyamides.By the heterologous expression of two E. coli lysine decarboxylases in C. reinhardtii, it was possible to significantly enhance the synthesis of cadaverine [190].The mutation of essential genes in the C. reinhardtii polyamine biosynthesis pathway identified ornithine decarboxylase 1 (ODC1) as a crucial regulator that controls the accumulation of putrescine.Subsequently, the authors overexpressed different ODCs, resulting in a significant increase in cellular putrescine levels, reaching a maximum yield of 200 mg/L [191] (Table 2).This achievement marks the first instance of microalgal bio-production of putrescine.
C. reinhardtii, Chlorella vulgaris, Dunaliella bardawil, Arthrospira platensis, Auxenochlorella protothecoides, and Euglena gracilis are among the very few microalgae recognized by the Food and Drug Administration as Generally Recognized as Safe (GRAS) organisms [177].This acknowledgment allows their use as a nutritional component in food, presenting new opportunities for the utilization of C. reinhardtii.Clinical studies on the human consumption of C. reinhardtii whole cells have demonstrated positive effects on gastrointestinal health and microbiota, showing that the intake of C. reinhardtii cells promotes microbiota eubiosis, reducing imbalances and improving the overall health of the intestine [192].The development of alternative plant-based products to substitute meat has led to the exploration of heme-containing proteins for their ability to provide a meat-like color and flavor.One such compound that can provide these qualities is protoporphyrin IX (PPIX) a crucial intermediate in the heme biosynthetic pathway.In this regard, engineered C. reinhardtii strains have been shown to overexpress PPIX [193].
Antioxidants are widely recognized for their beneficial impact on health and their crucial role in protecting cells from the harmful effects of free radicals.Chlamydomonas agloeformis has garnered attention due to its exceptionally high antioxidant capacities that surpass those of higher plants [194].Carotenoids are a diverse group of lipid-soluble pigments produced by plants and microorganisms, known for their benefits as vitamin precursors and antioxidants.Astaxanthin, a ketocarotenoid, is recognized as one of the most powerful natural antioxidants among carotenoids [195].Astaxanthin is currently primarily produced industrially from the microalgae Haematococcus pluvialis, with the crucial enzyme involved in its biosynthesis being β-carotene ketolase (BKT) [196].The synthetic redesign and overexpression of C. reinhardtii BKT has been shown to achieve Astaxanthin productivities of up to 4.3 mg/L/day, which is comparable to the results obtained with H. pluvialis [197] (Table 2).This production does not impair the growth or biomass productivity of C. reinhardtii, presenting a promising alternative to natural astaxanthin-producing algal strains.Furthermore, the accumulation of astaxanthin has led to enhanced high-light tolerance and increased biomass productivity [198].Blocking the expression of ATG1 and ATG8, genes involved in autophagy in C. reinhardtii, leads to a 2.3-times increase in carotenoid biosynthesis, indicating that autophagy does play a role in regulating carotenoid levels [199].
Chlamydomonas has been shown to be able to synthesize vitamins C, A, E, B 1 , B 7 , B 9 , and ergosterol, the precursor of vitamin D 2 [200].However, for most of these vitamins, the mechanisms regulating their synthesis to achieve overproduction have not been studied in detail.In C. reinhardtii, oxidative stress leads to a substantial increase in vitamin C levels [201].Omega-3 fatty acids play critical roles as nutrients and are extensively utilized in medicine.A comparison of C. reinhardtii with Chlorella and Spirulina revealed that C. reinhardtii contains superior amounts of omega-3 fatty acids, both in quality and quantity [202].Sulphated polysaccharides (SPs) are polymer chains containing one or more monosaccharide units that have been modified with sulfate groups.C. reinhardtii is capable of synthesizing SPs, which have been associated with several beneficial properties, including potent antioxidant and anticancer effects [203], antineurodegenerative effects [204], and antibiotic effects [205].
More than 40 therapeutic proteins, such as antibodies, enzymes, viral proteins, and hormones, among others, have been successfully expressed in C. reinhardtii [206].ICAM-1, a protein belonging to the immunoglobulin superfamily, was targeted for secretion into the extracellular media and was found to be fully active, suggesting that C. reinhardtii can produce mammalian proteins that are correctly folded and functional.Additionally, it achieved a concentration of up to 46.6 mg/L, marking the highest reported concentration of any recombinant protein in C. reinhardtii to date [207] (Table 2).The production of full-length spike protein, a crucial component for the infectivity of SARS-CoV-2, has been successfully achieved in C. reinhardtii as a secreted protein [208].This achievement is crucial as it offers a simpler and more economical platform for producing recombinant spike proteins in microalgae.

Conclusions and Future Perspective
Throughout this review, various studies conducted with Chlamydomonas on bioremediation and bioproduct production have been presented.These studies demonstrate diverse applications across different fields.Traditional Chlamydomonas biotechnology has focused on identifying productive strains through bioprospecting and enhancing productivity using forward genetics.However, significant advancements in Chlamydomonas bioproductivity require integrating these methods with emerging molecular genetics tools.The production of bioproducts from Chlamydomonas faces numerous challenges, even at the laboratory level, which become more pronounced on an industrial scale.The high production costs of Chlamydomonas, which surpass those of raw materials, render the process economically unviable at present.Addressing these challenges is essential for advancing these processes and fully realizing their industrial potential.
We believe that a critical area for future development, due to its significant industrial and environmental impact, would be the simultaneous integration of these two aspects.Biomass obtained from bioremediation should be utilized for producing specific bioproducts of interest.As highlighted in this review, there have been some initial attempts in this direction, although development is hindered by substantial challenges.Overcoming these obstacles, such as the presence of harmful residues like xenobiotics and heavy metals in the biomass, difficulties in scaling up biomass production, high energy demands, and concerns about contamination by bacteria, fungi, and viruses, represents the primary limitation to the industrial utilization of Chlamydomonas for bioremediation and subsequent biomass reuse.
Additionally, to achieve industrial application of the Chlamydomonas laboratory-level studies presented in this review, it is crucial to conduct an economic analysis of their feasibility, which has not yet been undertaken.The studies discussed here reflect significant efforts towards future improvements and optimizations aimed at mitigating these issues and promoting a circular economy approach.Such advancements would not only minimize waste and encourage material reuse but also generate substantial environmental, economic, and industrial benefits.

Figure 1 .
Figure 1.The Chlamydomonas-based phycoremediation process for wastewater treatment.The utilization of Chlamydomonas in wastewater phycoremediation is shown schematically, detailing the main cultivation methods, the employed mechanisms, and the various compounds that can be bioremediated.

Figure 1 .
Figure 1.The Chlamydomonas-based phycoremediation process for wastewater treatment.The utilization of Chlamydomonas in wastewater phycoremediation is shown schematically, detailing the main cultivation methods, the employed mechanisms, and the various compounds that can be bioremediated.

28 Figure 2 .
Figure 2.The process of Chlamydomonas bioproduct generation.Chlamydomonas cells can be cultivated in various conditions.After cultivation, the collected biomass and medium can then be processed as indicated to obtain the specified bioproducts (see Table2).

Table 1 .
Bioremediation characteristics and biomass production of different types of wastewaters using different strains of Chlamydomonas.Chemical oxygen demand (COD).

Table 2 .
Table summarizing the main characteristics of the different bioproducts generated by Chlamydomonas.